The invention relates to the field of immunology and, in particular, relates to vaccines against Chlamydia.
Chlamydia trachomatis is a species of the genus Chlamydiaceae, order Chlamydiales. C. trachomatis infects the epithelia of the conjunctivae and the genital tract, causing trachoma and a variety of sexually transmitted diseases (STDs) which can lead to, respectively, blindness or infertility. There are at least 15 serovars of C. trachomatis, of which A, B, and C are causative agents of trachoma, while serovars D, E, F, G, H, I, J, and K are the most common causative agents of chlamydial STDs. C. trachomatis infections are endemic throughout the world. Trachoma is the leading cause of preventable blindness in developing nations, and it is estimated that 600 million people suffer from trachoma worldwide, with as many as 10 million of them being blinded by the disease. In the United States there are an estimated 3 million cases per year of STDs caused by C. trachomatis. 
The pathogenesis of trachoma involves repeated ocular infections and the generation of a deleterious hypersensitivity response to chlamydial antigen(s) (refs 1 to 4 xe2x80x94Throughout this specification, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately following the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). The available evidence supports the hypothesis that both secretory IgA and cell-mediated immune responses are important components of protection. Ocular infection in a primate model induces rapid and persistent production of IgA in tears, whereas the presence of IgG in tears is transient, corresponding to the period of peak conjunctival inflammation (ref. 5). protective immunity following experimental ocular infection in a sub-human primate model is homotypic and resistance to ocular challenge correlates with the presence of serovar-specific antibodies in tears (refs. 1, 2, 6). Tears from infected humans neutralised the infectivity of homologous but not heterologous trachoma serovars for owl monkey eyes (ref. 7) whereas passive humoral immunization with antitrachoma antibodies was not protective (ref. 8). Several lines of evidence indicate the importance of cell-mediated responses in protection from or clearance of chlamydial infection. B-cell deficient mice can resolve infection, whereas nude mice become persistently infected. Adoptive transfer of at least some chlamydia-specific T-cell lines or clones can cure persistently infected nude mice, and this anti-chlamydial activity is probably a function of the ability of the T-cells to secrete interferonxcex3 (refs. 9 to 17).
past attempts to develop whole-cell vaccines against trachoma have actually potentiated disease by sensitizing vaccinees (refs. 1, 2). Sensitization has been determined to be elicited by a 57 kD stress response protein (SRP) (HSP60) present in all serovars of C. trachomatis. Repeated exposure to the 57 kD SRP can result in a delayed hypersensitivity reaction, causing the chronic inflammation commonly associated with chlamydial infections. Thus, an immunogenic preparation capable of inducing a strong and enduring mucosal neutralising antibody response and a strong cellular immune response without sensitizing the vaccinee would be useful (ref. 18).
A most promising candidate antigen for the development of a vaccine is the chlamydial major outer membrane protein (MOMP) (refs. 19 to 21). Other surface proteins and the surface lipopolysaccharide are also immunogenic, but the antibodies they induce have not been found to be protective (ref. 22, 23). The MOMP, which is the predominant surface protein, is an integral membrane protein with a mass of about 40 kDa which, with the exception of four variable domains (VDs) designated I, II, III, and IV, is highly conserved amongst serovars. The sequences of all four VDs have been determined for fifteen serovars (ref. 24, 25). Antibodies capable of neutralising chlamydial infectivity recognize the MOMP (ref. 26, 27, 28, 29). Epitopes to which MOMP-specific neutralising monoclonal antibodies bind have been mapped for several serovars (refs. 22, 23, 30, 31, 32, 33, 34), and represent important targets for the development of synthetic or subunit vaccines. The binding sites are contiguous sequences of six to eight amino acids located within VDs I or II, and IV, depending on the serovar. Subunit immunogens (e.g. isolated MOMP or synthetic peptides) containing MOMP epitopes can induce antibodies capable of recognising intact chlamydiae (ref. 26). However, conventionally administered subunit immunogens are generally poor inducers of mucosal immunity. It would be useful to formulate chlamydial antigens in such a way as to enhance their immunogenicity and to elicit both humoral and cell-mediated immune responses.
Immune stimulating complexes (ISCOMs) are cage-like structures formed from a mixture of saponins (or saponin derivatives), cholesterol and unsaturated fatty acids. The components of ISCOMs are held together by hydrophobic interactions, and consequently proteins which are naturally hydrophobic (such as MOMP) or which have been treated to expose or add hydrophobic residues can be efficiently incorporated into the ISCOMs as they form (ref. 35, 36, 37).
C. trachomatis naturally infects the mucosal surfaces of the eye and genital tract, and secretory IgA cellular responses are probably important components of protection. Consequently, it would be useful for a chlamydial vaccine to induce a mucosal immune response including both cellular and antibody components.
C. trachomatis infection may lead to serious disease. It would be advantageous to provide outer membrane antigen extracts of Chlamydia, including the major outer membrane protein of Chlamydia, particularly in substantially the native conformation for antigens in immunogenic preparations including vaccines, and immunogens and the generation of diagnostic reagents.
The present invention provides a novel immunogenic form of chlamydial MOMP which is useful in providing protection against chlamydial diseases, as well as methods of preparing such materials.
In accordance with one aspect of the invention, there is provided an immunogenic composition, comprising an outer membrane antigen extract (MAE) of a strain of Chlamydia, which may be Chlamydia trachomatis, and an immunostimulating complex (ISCOM).
The MAE may comprise the major outer membrane protein (MOMP) of the strain of Chlamydia. The MOMP may be in an oligomeric form and/or may be complexed with at least one other antigen of the strain of Chlamydia. Such oligomers and complexes may have a molecular weight of from about 45 to about 125 kDa. The procedure described herein for preparation of the MAE, specifically MOMP, produces material which is substantially free from the heat shock protein HSP60 of the strain of Chlamydia. The immunogenic composition provided herein may be in the form of the MEA incorporated into ISCOMs.
The immunogenic compositions provided herein may be employed, in accordance with another aspect of the invention, to protect a host against disease caused by a strain of Chlamydia by administering to the host an effective amount of the immunogenic composition. Such administration may be to a mucosal surface to produce a mucosal immune response. Alternatively, any other convenient means of administration may be employed to produce the desired immune response. The administration may be to the mucosal surface of the host by intranasal administration and may produce a genital tract immune response.
The present invention further includes a method of producing an outer membrane antigen extract of a strain of Chlamydia using a combination of two or more detergents including non-ionic detergents. Accordingly, in a further aspect of the invention, there is provided such a method which comprises:
detergent extracting elementary bodies (EBs) of the strain of Chlamydia in the presence of a reducing agent for disulphide bonds to solubilize cytoplasmic material away from outer membrane material;
separating the solubilized cytoplasmic material from the outer membrane materials,
detergent extracting the outer membrane material using at least two non-ionic detergents in the presence of a reducing agent for disulphide bonds to solubilize outer membrane antigens; and
separating the solubilized outer membrane antigens from residual unextracted membrane-associated material to provide the outer membrane antigen extract.
In one embodiment of this aspect of the invention, the at least two non-ionic detergents comprise a N-methyl glucamide non-ionic detergent which may be selected from heptanoyl-, octanoyl-, nonanoyl- and decanoyl-N-methyl glucamide, and a glucopyranoside non-ionic detergent, which may be selected from n-hexyl-xcex2-D-, n-heptyl-xcex2-D-, n-octyl-xcex1-D-, n-octyl-xcex2-D-, n-nonyl-xcex2-D-, n-decyl-xcex1-D- and n-decyl-xcex2-D-glucopyranoside. Such glucopyranosides are sometimes termed glucosides. The two non-ionic detergents may be employed in a weight ratio from about 1:10 to about 10:1. The use of the two detergents enables a high degree of recovery of outer membrane antigen which remain soluble at a wide range of temperature of storage.
The procedure described herein for the preparation of the outer membrane extract produces a novel Chlamydial antigen preparation. Accordingly, in an additional aspect of the present invention, there is provided a purified outer membrane antigen extract of a strain of Chlamydia in the form of an aqueous solution of antigen in the presence of at least two non-ionic detergents and a reducing agent.
In such composition, the purified outer membrane antigen extract comprises the major outer membrane protein (MOMP) of the strain of Chlamydia, particularly substantially in its native conformation. The MOMP usually comprises homooligomers and heterooligomers thereof, which may have the molecular weights from about 45 to about 125 kDa.
The provision of such novel purified materials enables there to be provided, in accordance with an additional aspect of the invention, a vaccine composition effective for protection against disease caused by a strain of Chlamydia, including Chlamydia trachomatis, comprising purified and non-denatured major outer membrane protein (MOMP) of the strain of Chlamydia substantially in its native conformation.
Such MOMP may be in the form of unaggregated homooligomers and heterooligomers. The vaccine composition may be in the form of immunostimulatory complexes (ISCOMs) incorporating the MOMP. The vaccine composition may further comprise at least one other chlamydial or non-chlamydial antigen.
The present invention further extends, in a further aspect of the invention, to a method for producing a vaccine against disease caused by a strain of Chlamydia, including Chlamydia trachomatis, comprising:
administering the vaccine composition provided herein to a test host to determine an amount and a frequency of administration thereof to confer protection against disease caused by the strain of Chlamydia; and
formulating the vaccine in a form suitable for administration to a treated host, which may be a human, in accordance with the determined amount and frequency of administration.